Mouse lactoferrin is expressed in a variety of tissues under different types of control. To understand how the molecular mechanisms govern the mode of lactoferrin expression, we isolated and characterized the 5' flanking region of the lactoferrin gene. By using previously cloned cDNA (T267), we isolated several clones containing lactoferrin gene fragments from mouse (129/J) genomic library. A clone (lambda-14) containing 7.5 kbp 5' flanking sequence of lactoferrin gene was identified. We sequenced completely a 3.0 ktp ECo R1/Hinc II DNA fragment containing a 2.6 kbp 5' flanking sequence. Analysis of this sequenced region revealed an estrogen-responsive element (ERE) consensus sequence overlapping with COUP binding element at -329 to -349 from the transcription initiation site. Synthetic oligonucleotides containing the mouse lactoferrin COUP/ERE elements (mERM) were cloned into reporter CAT gene and transiently transfected into human endometrial carcinoma RL95-2 cells to assess its hormone responsiveness. We found that the mERM confer estrogen action to both homologous and heterologous promoters in an orientation and distance independent manner. Nuclear proteins from DES-treated mouse uteri bound In vitro to mERM specifically as assessed by band-shift assay. Antibody to estrogen receptor and to COUP transcription factor interacted specifically with the mERM-protein complexes. The past year, this laboratory has also isolated the human lactoferrin cDNA (p1212). With this cDNA probe, we examined the polymorphism and methylation pattern of the lactoferrin gene in normal leukocytes, leukemic cells, and breast cancer. Msp1 Xba1 restriction enzyme fragment patterns demonstrated genetic polymorphism which occurred in DNA. The methylation patterns of DNA extracted from malignant cells was highly variable and generally less methylated than DNA extracted from normal WBCs. It is possible lactoferrin altered gene structure as well as altered regulation play a role in cancer development.